Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same

ABSTRACT

The disclosure provides devices, systems and methods for the generation of encapsulated reagents and the partitioning of encapsulated reagents for use in subsequent analyses and/or processing, such as in the field of biological analyses and characterization.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/977,804, filed Apr. 10, 2014, the full disclosure of which isincorporated herein by reference in its entirety for all purposes.

BACKGROUND

The field of life sciences has experienced dramatic advancement over thelast two decades. From the broad commercialization of products thatderive from recombinant deoxyribonucleic acid (DNA) technology, to thesimplification of research, development and diagnostics, enabled bycritical research tools, such as the polymerase chain reaction, nucleicacid array technologies, robust nucleic acid sequencing technologies,and more recently, the development and commercialization of highthroughput next generation sequencing technologies. All of theseimprovements have combined to advance the fields of biological research,medicine, diagnostics, agricultural biotechnology, and myriad otherrelated fields by leaps and bounds.

None of these technologies generally exist in a vacuum, but instead areintegrated into a broader workflow that includes upstream components ofsample gathering and preparation, to the downstream components of datagathering, deconvolution, interpretation and ultimately exploitation.Further, each of these advancements, while marking a big step forwardfor their fields, has tended to expose critical bottlenecks in theworkflows that must, themselves, evolve to fit the demands of the field.For example, genome sequencing is bounded on both ends by criticalworkflow issues, including, in many cases, complex and labor intensivesample preparation processes, just to be able to begin sequencingnucleic acids from sample materials. Likewise, once sequence data isobtained, there is a complex back-end informatics requirement in orderto deconvolve the sequence data into base calls, and then assemble thedetermined base sequences into contiguous sequence data, and ultimatelyalign that sequence data to whole genomes for a given organism.

One critical bottleneck for many of these technologies lies not in theirability to generate massive amounts of data, but in the ability to morespecifically attribute that data to a portion of a complex sample, or toa given sample among many multiplexed samples.

SUMMARY

Devices, methods and systems of the present disclosure provide solutionsto challenges in various fields, including the challenges describedabove. The present disclosure provides devices, systems and methods forthe generation of encapsulated reagents as well as multiplexedpartitions that include these encapsulated reagents for use in a varietyof applications.

The devices, systems and methods of the present disclosure employmicrofluidic systems in the generation of monodisperse populations ofmicrocapsules or beads that may have reagents such as biologicalreagents associated therewith. Also provided are devices, systems andmethods for selectively and controllably partitioning thesemicrocapsules or beads into droplets in emulsions for use in performingfurther reactions and/or analyses. Also provided are the variouscomponent parts of the devices and systems as well as interfacecomponents for facilitating interaction between such components.

An aspect of the disclosure provides a method for partitioningmicrocapsules. The method can include providing an aqueous fluidcomprising a suspension of microcapsules and flowing the aqueous fluidinto a droplet generation junction comprising a partitioning fluid toform a population of droplets of the aqueous fluid in the partitioningfluid. The flow rate of the aqueous fluid can be such that no more than50% of droplets of the population of droplets are unoccupied by amicrocapsule from the suspension of microcapsules.

In some embodiments, the flow rate is such that no more than 25% of thedroplets of the population of droplets are unoccupied by a microcapsule.In some embodiments, the flow rate is such that no more than 10% of thedroplets of the population of droplets are unoccupied by a microcapsule.In some embodiments, the flow rate is such that no more than 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2% or 1% of the population ofdroplets are unoccupied by a microcapsule.

In some embodiments, fewer than 25% of droplets of the population ofdroplets comprise more than one microcapsule. In some embodiments, fewerthan 20% of droplets of the population of droplets comprise more thanone microcapsule. In some embodiments, fewer than 15% of droplets of thepopulation of droplets comprise more than one microcapsule. In someembodiments, fewer than 10% of droplets of the population of dropletscomprise more than one microcapsule. In some embodiments, fewer than 5%of droplets of the population of droplets comprise more than onemicrocapsule.

In some embodiments, at least 80% of droplets of the population ofdroplets comprise a single microcapsule. In some embodiments, at least90% of droplets of the population of droplets comprise a singlemicrocapsule. In some embodiments, at least 95% of droplets of thepopulation of droplets comprise a single microcapsule. In someembodiments, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% ofdroplets of the population of droplets comprise a single microcapsule.

In some embodiments, the droplet generation junction can be in amicrofluidic channel network of a microfluidic device. In someembodiments, the microfluidic channel network can comprise a firstchannel segment fluidly connecting a source of microcapsules to thedroplet generation junction. The microfluidic channel network can alsocomprise a second channel segment connecting a source of partitioningfluid to the droplet generation junction, and a third channel segmentfluidly connected to the droplet generation junction providing an outletto the droplet generation junction.

In some embodiments, the flow rate can be provided by providing one ormore pressure differentials across the first and second channelsegments. In some embodiments, the first and/or second channel segmentscan have cross-sectional dimensions that provide the flow rate such thatno more than 50% of droplets of the population of droplets areunoccupied by a microcapsule from the suspension of microcapsules. Insome embodiments, the microfluidic channel network can further compriseone or more flow controlling structures within the first channel segmentthat provide the flow rate.

In some embodiments, the microcapsules of the suspension ofmicrocapsules have a mean cross-sectional dimension and a coefficient ofvariation in cross-sectional dimension of no greater than 10%. In someembodiments, the microcapsules of the suspension of microcapsules have amean cross-sectional dimension and a coefficient of variation incross-sectional dimension of no greater than 10%, 8%, 6%, 4%, 2% or 1%.

An additional aspect of the disclosure provides a method forpartitioning microcapsules. The method can include flowing an aqueousfluid comprising a suspension of microcapsules into a droplet generationjunction comprising a partitioning fluid. During a window of dropletgeneration, the microcapsules can be flowing into the droplet generationjunction at a frequency that varies less than 30%. The method can alsoinclude partitioning the microcapsules in the partitioning fluid duringthe window of droplet generation. In some embodiments, the frequency isgreater than 50 Hz. In some embodiments, the frequency is greater than500 Hz. In some embodiments, the frequency is greater than 1000 Hz. Insome embodiments, the frequency is greater than 50 Hz, 100 Hz, 250 Hz,500 Hz, 750 Hz, 1000 Hz, 1250 Hz, 1500 Hz, 1750 Hz or 2000 Hz.

In some embodiments, during the window of droplet generation, themicrocapsules flow into the droplet generation junction at a frequencythat varies less than 20%. In some embodiments, during the window ofdroplet generation, the microcapsules flow into the droplet generationjunction at a frequency that varies less than 10%. In some embodiments,during the window of droplet generation, the microcapsules flow into thedroplet generation junction at a frequency that varies less than 5%. Insome embodiments, during the window of droplet generation, themicrocapsules flow in the droplet generation junction at a frequencythat varies less than 30%, 25%, 20%, 15%, 10%, 5%, 2% or 1%.

In some embodiments, flowing the aqueous fluid comprising the suspensionof microcapsules in the droplet generation junction comprising apartitioning fluid can comprise flowing the aqueous fluid through amicrofluidic channel fluidly connected to the droplet generationjunction. The microfluidic channel can include a region that regulatesthe flow (e.g., flow rate) of the microcapsules.

An additional aspect of the disclosure provides a method for producingmicrocapsules. The method can include providing a gel precursor in anaqueous fluid and flowing the aqueous fluid having the gel precursorthrough a fluid conduit that is fluidly connected to a dropletgeneration junction comprising a partitioning fluid. The partitioningfluid can comprise a gel activation agent. The method can also includeforming droplets of the aqueous fluid in the partitioning fluid, where,within the droplets, the gel activation agent contacts the gel precursorto form gel microcapsules. In some embodiments, the aqueous fluid canalso comprise a biological molecule, where, for example, the biologicalmolecule can become entrained in the gel microcapsules.

An additional aspect of the disclosure provides a method forpartitioning microcapsules. The method can include flowing an aqueousfluid comprising a suspension of a monodisperse population ofmicrocapsules into a droplet generation junction. The monodispersepopulation can have a mean cross-sectional dimension and a coefficientof variation in cross-sectional dimension of no greater than 10%. Themethod can also include introducing a partitioning fluid into thedroplet generation junction and separating the aqueous fluid intodroplets within the partitioning fluid, where the droplets contain oneor more microcapsules.

An additional aspect of the disclosure provides a microfluidic system.The microfluidic system can include a microfluidic channel networkcomprising at least first, second and third channel segments in fluidcommunication with a droplet generation junction. The first channelsegment can be fluidly connected to a first fluid source that comprisesa first fluid that comprises an aqueous fluid. The aqueous fluid cancomprise a plurality of microcapsules disposed therein. Moreover, thesecond channel segment can be fluidly connected to a second fluid sourcethat comprises a second fluid that is immiscible with the aqueous fluid.The microfluidic system can also include a flow control system connectedto the microfluidic channel network. The flow control system can subjectthe first fluid and second fluid to flow into the droplet generationjunction to generate droplets that comprise microcapsules; and cansubject the droplets to flow into the third channel segment such that atleast 75% of the droplets comprise at least one microcapsule and fewerthan 25% of the droplets comprise more than one microcapsule.

An additional aspect of the disclosure provides a microfluidic system.The microfluidic system can include a microfluidic channel network. Themicrofluidic channel network can comprise a first channel segmentcoupled to a source of a first aqueous fluid that comprises a suspensionof microcapsules; at least one second channel segment coupled to asource of a second aqueous fluid, the first and second channel segmentsin fluid communication at a first junction that brings the first aqueousfluid in contact with the second aqueous fluid; and a third channelsegment coupled to the first junction and intersecting at least onefourth channel segment at a second junction. The at least one fourthchannel segment can be coupled to a source of a fluid that is immisciblewith the first and second aqueous fluids. Moreover, the second junctioncan partition the first and second aqueous fluids into droplets withinthe fluid. The microfluidic system can also include a flow controlsystem operably coupled to the microfluidic channel network. The flowcontrol system can subject the first, second and third fluids to flowthrough the microfluidic channel network to form droplets comprising thefirst and second aqueous fluids in the fluid, at a frequency of at least50 Hz and that varies less than 20%.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C provide schematic illustrations of example partitionor droplet generating fluidic channel junctions.

FIG. 2 schematically illustrates a simple, example fluidic channelarchitecture for partitioning microcapsules and other fluids intodroplets in a water-in-oil emulsion.

FIGS. 3A and 3B schematically illustrate an example fluid channelarchitecture for partitioning encapsulated reagents into droplets in anemulsion.

FIG. 4 schematically illustrates an example channel network andmicrofluidic device useful in partitioning encapsulated reagents.

FIG. 5 schematically illustrates a side view of an example reservoirstructure for enhancing manipulation of microcapsule compositions withinfluidic devices.

FIGS. 6A and 6B illustrates an example microcapsule flow regulatingstructure.

FIGS. 7A and 7B schematically illustrates an example of interfacingfluid containing vessels with a fluid reservoir on a device.

DETAILED DESCRIPTION I. General

The present disclosure provides devices, systems and methods that areparticularly useful in managing complex samples for analysis using highthroughput analytical systems, including, for example, high throughputnucleic acid analysis systems, such as nucleic acid arrays, nucleic acidsequencing systems, nucleic acid amplification and quantitation systems,or the like. In particular, the devices, systems and methods describedherein are particularly useful in providing encapsulated reagents orreagent systems, and co-partitioning these reagents with samplecomponents for further reaction and/or analysis. This co-partitioning ofreagents and sample components can be used, for example, in reducing thecomplexity of the sample material by segregating portions of the sampleto different partitions. Further, by also segregating reagents, one cansubject each sample portion to a different reaction, including forexample, the application of unique identifiers to different samplecomponents, e.g., attachment of a discrete barcode or tagging reagentsto the discrete sample components.

Particularly elegant examples of these co-partitioning approaches aredescribed in Published International Patent Application No.WO2014/028537, and U.S. patent application Ser. Nos. 14/104,650 (filedDec. 12, 2013), 14/175,935 (filed Feb. 7, 2014), 14/175,973 (filed Feb.7, 2014), and 61/937,344 (filed Feb. 7, 2014), the full disclosures ofeach of which are incorporated herein by reference in their entirety forall purposes.

By way of example, one particularly elegant approach provides a polymermicrocapsule composition that includes nucleic acid barcode sequencesbound to the microcapsule, where the barcodes associated with a givenmicrocapsule have substantially the same sequence of nucleotides, butwhere different discrete microcapsules will have different barcodesequences associated with such microcapsules. Each of thesemicrocapsules is then contacted with a portion of a sample fluid, suchas a sample fluid that includes a template nucleic acid from a samplematerial. The mixture of sample material including the template nucleicacid and the microcapsule is then partitioned into a small volume, suchas a droplet in a water in oil emulsion, such that the microcapsule anda portion of the sample material are contained within the same droplet.In addition to controlling the droplet generation process to provide adesired number of microcapsules in a given partition, the samplematerial and emulsion process also may be controlled to provide for adesired amount of sample material, e.g., sample nucleic acid material,within each partition, e.g., to provide a single template molecule or adesired level of genome coverage within a given partition, or otherdesired level of sample materials.

Within the partition, the barcode sequence is reacted with the samplematerial, e.g., the template nucleic acid to effectively tag the samplematerial or a portion thereof. For example, by reacting the barcodesequence with the template, e.g., through amplification of the templatesequence using the barcode sequence as an extension primer, one caneffectively “attach” the barcode sequence to the replicated or amplifiedtemplate. Similarly, replication of the extended primer produces acomplement of the template along with a complement to the barcode,again, effectively attaching the barcode to the template. The presenceor attachment of the barcode sequence, or its complement, on or to theamplified template molecule, or its complement, then allows some levelof attribution of sequence reads that include that barcode to the sameportion of sample material, e.g., the same template molecule or the samesample components, that was originally allocated to that partition.

In many cases, the molecule that includes the barcode sequence orsequences may also include functional elements that are used insubsequent processing of the amplified template sequences. Thesefunctional sequences include, for example, primer sequences (e.g.,targeted or universal), primer recognition sequences, sequences that canform secondary structures, either within the sequence, or uponreplication of the sequence, enrichment sequences, e.g., that are usedas affinity purification sequences, immobilization sequences, probesequences, reverse complement or hairpin sequences, or any of a varietyof other functional sequences.

There are a wide variety of other high-value applications for suchpartitioning and barcoding or tagging processes. The present disclosureadvantageously provides devices, systems and methods that can greatlyfacilitate the generation of such partitioned compositions or componentsthereof.

II. Fluidic Systems for Producing Encapsulated Reagents and PartitionedReactions

The present disclosure provides improved fluidic systems, andparticularly improved microfluidic systems, that are useful for both thegeneration of encapsulated reagents, as well as in the partitioning ofthose encapsulated reagents for use in subsequent reactions and/oranalyses. As used herein, microfluidic systems typically denote fluidicsystems that employ one or more fluid conduits, channels, chambers, orthe like that include one or more interior cross-sectional dimensions,e.g., depth, length or width, that are less than 1000 microns, less than200 microns, less than 100 microns, and in some cases, less than about50 microns, or even less than about 20 microns. In some cases, one ormore cross-sectional dimensions may be about 20 microns or less or 10microns or less. Typically, these microfluidic channels or chambers willhave at least one cross-sectional dimension of between about 1 and about100 microns.

As will be appreciated, reference to encapsulated reagents is notintended to limit the scope of such reagents to completely enclosedcapsules, but is intended to reflect any of a variety of methods ofassociating reagents with a given particle, bead, or other solid orsemi-solid particle phase. In particular, encapsulation generally refersto the entrainment or other attachment, coupling, or association of aparticular species with a solid or semi-solid particle, bead, enclosure,partition or droplet, and is not limited to compositions in which thespecies is entirely or partially enclosed within a larger structure.

In some aspects, encapsulated reagents are associated with microcapsulesthat are generally spherical in shape, although they may be elongated,plug shaped, or otherwise vary in their specific shape. In some cases,microcapsules will have one or more cross-sectional dimensions that areless than 200 microns, less than 150 microns, or less than about 100microns. In some cases, microcapsules of the present disclosure have oneor more cross-sectional dimensions that are between about 10 and about200 microns, between about 20 and 150 microns, between about 30 and 125microns, in many cases between about 40 and about 100 microns, and stillother cases, between about 50 and about 75 microns.

While the dimensions of the microcapsules can be an importantconsideration, in many applications the variability in those dimensionsis also an important consideration. In particular, for example, thetransport of a microcapsule through a microfluidic system can besignificantly impacted by the size of that microcapsule. For examplesimple flow resistance may be greater for much larger microcapsules thanfor smaller microcapsules. Similarly, propensity for clogging may begreater for larger microcapsules than for smaller microcapsules. Ineither event, flow rates of microcapsules through a microfluidic systemmay be greatly impacted by the size of the microcapsule. Accordingly, incertain aspects, the microcapsules of described herein, will be providedas a population of microcapsules having substantially monodispersecross-sectional dimensions. In terms of cross-sectional dimensions, thephrase substantially monodisperse refers to a population that deviates(e.g., expressed as a coefficient of variation and stated as apercentage) from the mean cross-sectional dimension by no more than 50%,no more than 40%, no more than 30%, no more than 20%, or in some cases,no more than 10%.

Whether in the context of generating microcapsules for use inentrainment or encapsulation of reagents, or in the partitioning ofaqueous fluids within non-aqueous droplets, the devices and systems ofthe present disclosure can employ a similar architecture. In asimplified example, this architecture may include a first channelsegment that is fluidly connected to a first junction that fluidlyconnects the first channel segment with a second channel segment and athird channel segment. The second channel segment delivers to thejunction a second fluid that is immiscible with the first aqueous fluid,such as an oil, that allows for the formation of aqueous droplets withinthe stream of immiscible fluid. This second fluid may be referred toherein as the dispersion fluid, partitioning fluid or the like. The flowof the first and second fluids through the junction and into the thirdchannel segment is controlled such that droplets of the first fluid aredispensed into a flowing stream of the second fluid within the thirdchannel segment. A variety of modifications to this basic structure areavailable to better control droplet formation and to bring in additionalfluid streams. As used herein, the control of fluid flows encompassesboth active control of fluid flows through the application of greater orlesser driving forces to cause that fluid flow. Additionally, flows maybe controlled in whole or in part, by controlling the flowcharacteristics of one or more of the fluids and/or the conduits throughwhich they are flowing. For example, fluid flow may be controlled byproviding higher flow resistance within a conduit, e.g., throughproviding a higher viscosity, narrower conduit dimension, or providinglarger or smaller microcapsules within a fluid stream, or anycombination of the foregoing. In some cases, control is imparted throughseveral of controlled driving force, controlled conduit dimensions, andcontrolled fluid properties, e.g., viscosity or particle composition.

FIG. 1A provides a schematic illustration of an exemplary basic channelarchitecture for generating droplets in a channel. As shown, firstchannel segment 102, second channel segment 104, third channel segment106 and fourth channel segment 108 are all provided in fluidcommunication at first junction 110. FIG. 1B schematically illustratesdroplet formation within the channel architecture of FIG. 1A.

As shown, a first aqueous fluid 112 is flowed through channel segment102 toward junction 110. A second fluid 114 that is immiscible with thefirst fluid 112 is flowed into junction 110 via each of channel segments104 and 106, and into fourth channel segment 108. As the aqueous firstfluid 112 reaches the junction 110, it is pinched by the flow of thesecond fluid 114 from channel segments 104 and 106, and individualdroplets 116 of the aqueous first fluid 112 are dispensed into fourthchannel segment 108. In some cases, a portion of the fourth channelsegment 108 proximal to the junction 110 may be provided with a reducedcross-section (not shown) as compared to the junction and/or channelsegments 102, 104 and 106 to facilitate droplet formation within thefourth channel segment 108.

As discussed in greater detail below, additional channel segments may beprovided either upstream, downstream or both, of junction 110, in any ofchannel segments 102, 104, 106 or 108, to allow for the delivery ofadditional fluids into either the aqueous first fluid stream in segment102, e.g., additional reagents, buffers, or the like, the partitioningfluid in segments 104 and/or 106, or the droplet containing stream inchannel segment 108.

As will be appreciated, this basic channel architecture is widely usefulin both generation of microcapsules for encapsulation of reagents, aswell as in the ultimate partitioning of those encapsulated regents withother materials.

In one particular example and with reference to FIGS. 1A and 1B, above,a first aqueous solution of polymer precursor material may betransported along channel segment 102 into junction 110 as the aqueousfluid 112, while a second fluid 114 that is immiscible with the polymerprecursor is delivered to the junction 110 from channel segments 104 and106 to create discrete droplets of the polymer precursor materialflowing into channel segment 108. In some aspects, this second fluid 114comprises an oil, such as a fluorinated oil, that includes afluorosurfactant for stabilizing the resulting droplets, e.g.,inhibiting subsequent coalescence of the resulting droplets. Examples ofparticularly useful partitioning fluids and fluorosurfactants aredescribed for example, in U.S. Patent Application No. 2010-0105112, thefull disclosure of which is hereby incorporated herein by reference inits entirety for all purposes. Polymer precursor materials may includeone or more of polymerizable monomers, linear polymers, or other

In preparing gel microcapsules, an activation agent may also be combinedwith the aqueous stream 112 from channel 102. In some aspects, thisactivation agent is disposed within the second fluid streams 114 in oneor more of channels 104 and 106, allowing for the simultaneous formationof droplets and commencement of a reaction to create the desiredmicrocapsules 116. For example, in the case where the polymer precursormaterial comprises a linear polymer material, e.g., a linearpolyacrylamide, PEG, or other linear polymeric material, the activationagent may comprise a cross-linking agent, or a chemical that activates across-linking agent within the first stream. Likewise, for polymerprecursors that comprise polymerizable monomers, the activation agentmay comprise a polymerization initiator. For example, in certain cases,where the polymer precursor comprises a mixture of acrylamide monomerwith a N,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams in channel segments 104 and 106, which initiates thecopolymerization of the acrylamide and BAC into a cross-linked polymernetwork or, hydrogel.

Upon contact of the second fluid stream 114 with the first fluid stream112 at junction 110 in the formation of droplets, the TEMED may diffusefrom the second fluid 114 into the aqueous first fluid 112 comprisingthe linear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets, resulting in the formation of thegel, e.g., hydrogel, microcapsules 116, as solid or semi-solid beads orparticles.

Although described in terms of polyacrylamide encapsulation, other‘activatable’ encapsulation compositions may also be employed in thecontext of the present disclosure. For example, formation of alginatedroplets followed by exposure to divalent metal ions, e.g., Ca2+, can beused as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling, e.g., upon cooling, or the like.

In accordance with some aspects of the present disclosure one or morereagents may be associated with the microcapsule at the time of itsformation. In particular, one or more reagents may be associated with aprecursor reagent to the polymer matrix that makes up the microcapsulee.g., the linear polymer, such that the reagent(s) will be entrainedwithin or otherwise associated with the formed microcapsule. Forexample, the reagent(s) may be coupled to a linear polymer material thatis cross-linked into a microcapsule using the processes describedherein, resulting in the reagents being coupled to the formed andcross-linked gel microcapsule. Alternatively, the reagent may becombined with the polymer precursor that includes active binding sitesthat interact with the reagent, either in the precursor stream or in themicrocapsule after formation. In still other aspects, as with thecross-linking activation agent described elsewhere herein, an activatormay also be contacted with the polymer precursor or formed microcapsulethat activates sites on the polymer matrix of the microcapsule to whichthe reagent components may associate, covalently or non-covalently.

Reagents to be incorporated into the microcapsule may include any of avariety of different reagents or other components useful in the ultimateuse of the microcapsule, e.g., an analytical reaction. Such reagents mayinclude labeling groups (e.g., fluorescent dye molecules, FRET pairs,fluorescent nanoparticles, fluorescent proteins, mass labels,electrochemical labels or the like). These reagents may includebiological or biochemical reagents, such as nucleic acids, nucleic acidanalogues, nucleic acid mimetics, polynucleotides or analogues,oligonucleotides or analogues, enzymes, substrates, antibodies orantibody fragments, antigens, epitopes, receptors, and receptor bindingcomponents, proteins, polypeptides, amino acids, polysaccharides, orvirtually any type of biochemical reagent useful in any of a widevariety of analyses. Likewise, compounds that act upon biological orbiochemical systems are also envisioned for inclusion in suchmicrocapsules, e.g., small molecule pharmaceutically active compounds,radiological compounds, inhibitors and or initiators of biological orbiochemical compounds, chemical library compounds, or the like. Incertain examples, these reagents may include any of a wide of variety ofdifferent reagents that are applicable to desired reactions to becarried out within the ultimately created partition, such as nucleicacid replication reagents (e.g., primers, polymerases, nucleotides ornucleotide analogues, buffers, co-factors, or the like), specificbinding groups (e.g., receptors, antibodies or antibody fragments,binding peptides), or any other reagents (e.g., enzymes, substrates,catalysts/initiators, substrates, inhibitors, or the like).

In one example, a polynucleotide having an acrydite moiety is providedwithin the aqueous fluid, where the polynucleotide is coupled to thepolymer precursor prior to its cross-linking into a bead as describedherein. This polynucleotide may comprise one or more functional nucleicacid sequences, such as primer sequences, attachment sequences, ligationsequences or barcode sequences. See, e.g., U.S. Patent Application No.61/937,344, which is entirely incorporated herein by reference.

Once created, the microcapsules may be collected, e.g., from a reservoiror other outlet at the end of channel segment 108. The collectedmicrocapsules may then be washed to remove crosslinking agent,non-crosslinked polymer, emulsion oil and surfactant, any othernon-coupled reagents, out-sized microcapsules or portions thereof, aswell as any other contaminants imparted to the microcapsules duringtheir creation that may potentially interfere with the use of themethods and systems described herein. In some aspects, the microcapsuleswill comprise substantially pure microcapsule compositions. Bysubstantially pure microcapsule compositions is meant that themonodisperse populations of microcapsules, as described above, and theirassociated desired buffer and reagents will make up at least 90% of thecomposition, at least 95% of the composition, at least 99% of thecomposition, and in many cases at least 99.9% of the composition. Oncewashed, these microcapsules may be re-suspended in an aqueous solution,e.g., a buffer and/or one or more selected reagents, for use insubsequent processing. In accordance with the above, a variety ofdifferent wash protocols may be used in series or in the alternative ingenerating the substantially pure microcapsules described above. By wayof example, in some cases, the wash may comprise a simple bufferexchange wash where the microcapsules are separated from theirsupporting liquid, e.g., through settling, centrifugation, filtration,or the like, and then re-suspended in a new buffer solution that may ormay not be the same buffer as was originally containing themicrocapsules. This type of wash may be repeated multiple times toremove free contaminants from the microcapsules. In alternative oradditional wash steps, a more stringent washing process may be employedto remove certain bound species from the microcapsules. For example,where a microcapsule comprises nucleic acid, protein or other associatedreagents, a denaturing wash step may be employed to remove additionalbound excess proteins, nucleic acids or the like. For example, in somecases, the microcapsules may be washed with chaotropic agents, such asurea, at elevated temperatures to remove other non-covalently boundspecies, e.g., hybridized nucleic acids, etc. In still other aspects,wash steps may be combined with extractive techniques, in order toremove species that may be entrained within the interior of themicrocapsules. For example, in some cases, these extractive processesmay include electroelution, osmotic elution or other techniques to drawnon-covalently bound species from within microcapsules.

In many cases, the substantially pure microcapsule compositions aresubstantially free from aggregated microcapsules, e.g., two, three, fouror more microcapsules adhered together. Separation of aggregatedmicrocapsules may be carried out through a variety of methods, includingfor example, size or flow based separation techniques, e.g., filtration.

Although described with reference to the channel architecture shown inFIGS. 1A and 1B, it will be appreciated that variations of thesestructures and architectures may be practiced within the scope of thepresent disclosure. For example, in some cases, the interface of theaqueous stream with the partitioning fluid may differ from the specificarchitectures described above. In particular, as shown in FIG. 1A, theintersection of channel segment 112 with channel segments 104 and 106provides an interface between the aqueous fluid flow in channel segment102 and the partitioning fluid. The droplets are formed as the aqueousfluid is pushed into and through that interface into channel segment108. In some cases, however, the interface may be presented within anopen space or chamber or channel segment manifold within a fluidicdevice, such that the interface exists as a “wall” of partitioningfluid. An example of this type of droplet generation junction isillustrated in FIG. 1C. As shown, a first channel segment 122 is fluidlyconnected to a fluid manifold 132 that forms part of the dropletgeneration junction. The manifold 132 is structured as a larger openchamber, i.e., larger than the first channel segment, with a dropletdispensing channel 134 exiting the manifold through which formeddroplets 138 are expelled through dispensing channel or aperture 134into channel segment 136. In some cases, additional side channelsegments 124 and 126 are also provided fluidly connected to the manifold132, as are channel segments 128 and 130. In operation, a first aqueousfluid (e.g., the aqueous polymer precursor fluid as described withreference to FIG. 1B, or the microcapsule containing aqueous fluiddescribed with reference to FIG. 3B, below) is flowed into the manifold132. An immiscible fluid is introduced into the manifold through sidechannels 128 and 130. Within the manifold 132, the immiscible fluidforms an interface that traverses the manifold 132 to the dropletdispensing port (shown as the dashed lines extending from channelsegments 128 and 130 to dispensing channel 134). In some cases,additional aqueous fluids are introduced into the manifold through sidechannels 124 and 126. As the fluids flow through the droplet dispensingchannel 134, the aggregate aqueous fluids, i.e., that from channelsegment 122 and in some cases from segments 124 and 126, are surroundedby the immiscible fluid from channel segments 128 and 130 and expelledthrough dispensing channel segment 134 into channel segment 136 asdroplets 138 of aqueous fluids within an immiscible fluid emulsion. Aswill be appreciated, controlling the rate of droplet formation, as wellas the relative volumes of fluids combined in droplets within thesetypes of structures is accomplished through many of the same mechanismsdescribed above for basic channel intersections. In particular,controlled flow may be achieved through a number of mechanisms,including, for example, controlling the flow rates of the fluids beingintroduced into the manifold, controlling the geometry of the channelsas they enter the manifold 132, e.g., channel shape, dimensions (depthand/or width), intersection contours and structure, and setback from themanifold as compared to other channels.

Additionally, although illustrated in FIG. 1A as a single interface fordroplet generation, it will be appreciated that the devices and systemsof the present disclosure will typically comprise multiplexed dropletgenerating interfaces in order to increase the throughput at which onecan produce droplets for microcapsule formation or for partitioning ofmicrocapsules, as described elsewhere herein. For example, a device orsystem of the present disclosure may include multiple duplicate channelnetworks of the architectures shown in FIGS. 1A and/or 1C. Further, forsuch multiplexed devices or systems, some of the various channelsegments within the duplicate channel networks may have common fluidsources in terms of a common reservoir or a common channel or channelmanifold, or may feed to a common outlet or reservoir. Likewise, in thecase of alternate architectures, multiple aqueous fluid feed channelsegments may be provided in communication with the partitioning fluidchamber.

FIG. 2 schematically illustrates a microfluidic device or device modulefor producing the microcapsules described above. As shown, themicrofluidic device typically includes a body structure 200 thatincludes within its interior portion, a channel network that includeschannels segments 202, 204, 206 and 208. These channel segments allcommunicate with a common channel junction 210. The device bodystructure also includes reagent reservoirs 212 and 214. As shown,reagent reservoir 212 is fluidly coupled to channel segment 202, whilereagent reservoir 214 is fluidly coupled to channel segments 204 and206. A third outlet reservoir is shown as reservoir 216, which isprovided in fluid communication with channel segment 208. As will beappreciated, the aqueous polymer gel precursor may be provided inreservoir 212, while the partitioning fluid and activating agent areprovided in reservoir 214. Flow of these fluids through junction 210,creates the microcapsules as described above, which flow into and areharvested from reservoir 216.

These microfluidic devices or device modules may be fabricated in any ofa variety of conventional ways. For example, in some cases the devicescomprise layered structures, where a first layer includes a planarsurface into which is disposed a series if channels or grooves thatcorrespond to the channel network in the finished device. A second layerincludes a planar surface on one side, and a series of reservoirsdefined on the opposing surface, where the reservoirs communicate aspassages through to the planar layer, such that when the planar surfaceof the second layer is mated with the planar surface of the first layer,the reservoirs defined in the second layer are positioned in fluidcommunication with the termini of the channel segments on the firstlayer. Alternatively, both the reservoirs and the connected channelstructures may be fabricated into a single part, where the reservoirsare provided upon a first surface of the structure, with the aperturesof the reservoirs extending through to the opposing surface of thestructure. The channel network is fabricated as a series of grooves andfeatures in this second surface. A thin laminating layer is thenprovided over the second surface to seal, and provide the final wall ofthe channel network, and the bottom surface of the reservoirs.

These layered structures may be fabricated in whole or in part frompolymeric materials, such as polyethylene or polyethylene derivatives,such as cyclic olefin copolymers (COC), polymethylmethacrylate (PMMA),polydimethylsiloxane (PDMS), polycarbonate, polystyrene, polypropylene,or the like, or they may be fabricated in whole or in part frominorganic materials, such as silicon, or other silica based materials,e.g., glass, quartz, fused silica, borosilicate glass, or the like.

Polymeric device components may be fabricated using any of a number ofprocesses including embossing techniques, micromachining, e.g., lasermachining, or in some aspects injection molding of the layer componentsthat include the defined channel structures as well as other structures,e.g., reservoirs, integrated functional components, etc. In someaspects, the structure comprising the reservoirs and channel structuresmay be fabricated using, e.g., injection molding techniques to producepolymeric structures. In such cases, a laminating layer may be adheredto the molded structured part through readily available methods,including thermal lamination, solvent based lamination, sonic welding,or the like.

As will be appreciated, structures comprised of inorganic materials alsomay be fabricated using known techniques. For example, channel and otherstructures may be micro-machined into surfaces or etched into thesurfaces using standard photolithographic techniques. In some aspects,the microfluidic devices or components thereof may be fabricated usingthree-dimensional printing techniques to fabricate the channel or otherstructures of the devices and/or their discrete components.

As noted previously, the above-described channel architectures may alsobe readily employed in the partitioning of the above describedmicrocapsules, e.g., comprising the encapsulated reagents, withindroplets created in an immiscible fluid, such as in a “water-in-oil”(WO) emulsion system, where an aqueous solution, and particularly, anaqueous solution that includes the encapsulated reagents describedherein, is dispersed as partitioned droplets within an immiscibledispersion or partitioning fluid, such as an immiscible oil.

FIG. 3 schematically illustrates the partitioning of encapsulatedreagents. As shown, and with reference to the fluidic architecture shownin FIG. 1A, a first aqueous fluid that includes the beads encapsulatingat least a first reagent is flowed through channel segment 102 intochannel junction 110. The dispersion fluid is flowed into junction 110from side channel segments 104 and 106. The aqueous fluid is thenpartitioned into droplets within the flowing stream of dispersion fluid,with individual droplets including the encapsulated reagents, and insome cases, containing only a single reagent bead or capsule.

The above-described channel architecture is included within an exampleof a channel system shown in FIG. 3A, for partitioning microcapsules,including, e.g., encapsulated reagents, with sample materials into, forexample, a water-in-oil emulsion system. As shown, a first channelsegment 302 is shown fluidly connected to channel segments 304, 306 and308 at first channel junction 310. Fourth channel segment 308 fluidlyconnects first channel junction 310 to second channel junction 322 thatis also fluidly coupled to channel segments 324, 326 and 328.

In the context of partitioning encapsulated reagents, the channel systemof FIG. 3A is shown in FIG. 3B. As shown, a first stream of a firstaqueous fluid 312 containing microcapsules 350 (e.g., such asmicrocapsules prepared as described above), beads or the like, that mayinclude encapsulated reagents, are flowed through channel segment 302into channel junction 310. Additional streams of second aqueous fluids352 and 354 are introduced into channel junction 310 from channelsegments 304 and 306 to join the first aqueous fluid 312 containing themicrocapsules 350. The aqueous fluids added through each of channelsegments 304 and 306 may be the same as or different from each other andthe fluid portion of aqueous stream 312. As will be appreciated, thevarious channel segments will typically be fluidly coupled to sources ofthe fluids that are to be flowed through those channel segments. Suchfluid sources may include reservoirs integrated within a device orinterfaced with a device, or may include other interfaces with otherfluidic systems, e.g., syringes, pumps, fluidic networks or the like, orinterfaced with external reservoirs, e.g., external fluid accessionsystems for drawing fluids from tubes, vials, wells, or the like, oreven external processing systems, e.g., amplification systems, samplematerial extraction systems, filtration systems, separation systems,liquid chromatography systems, or the like.

In some aspects, the additional aqueous fluids added through sidechannels 304 and 306 may include sample materials that are to bepartitioned along with the encapsulated reagents included within themicrocapsules. For example, the second aqueous fluid may include samplenucleic acids that may be partitioned into separate droplets along withthe reagents included with the microcapsules, such as barcode sequences,functional sequences and the like. Additional reagents may also be addedin the second aqueous fluids. In some cases, e.g., where theencapsulated reagents are to be employed in nucleic acid replication orsynthesis reactions, the additional fluids may include reagents for suchreactions, such as DNA polymerase enzyme(s), primer sequences,nucleotides or nucleotide analogues, reaction co-factors, buffers andthe like, as well as any of a variety of other reagents, e.g., dyes,labels, chelators, inhibitors, initiators, substrates, etc.

In some cases, the reagents that are added may include reagents thatstimulate release of the encapsulated reagents into the resultingdroplets. For example, in some cases, the reagents may be associatedwith the microcapsule through a disulfide linkage or other chemicallycleavable linkage, or the microcapsules may be structurally heldtogether by disulfide crosslinking, or other chemically cleavablecross-linkers. As such, addition of a reducing agent, such asdithiothreitol (DTT) can result in the eventual release of the reagentson the microcapsules, either through direct release or throughdissolution of the microcapsule, or both (See, e.g., U.S. PatentApplication No. 61/940,318, filed Feb. 14, 2014, the full disclosure ofwhich is incorporated herein by reference in its entirety for allpurposes). Alternatively or additionally, other cleavable linkages maybe used to crosslink microcapsules. Examples of such linkages include,e.g., photocleavable or chemically cleavable linkages or cross-linkers.

The combined aqueous stream, e.g., from fluids 312, 352 and 354, flowsthrough channel segment 308 into channel junction 322. A third fluid 314that is immiscible with the combined aqueous stream flowing from channelsegment 308 is introduced into channel junction 312 from each of channelsegments 324 and 326 to form droplets 356 that include the microcapsules350, as well as some amount of the combined aqueous fluids. In manycases, this third, immiscible fluid includes an oil, such as afluorinated oil containing a fluorosurfactant, as described above thatis suitable for forming water-in-oil emulsions with stabilized resultingdroplets. Other suitable emulsion systems may in some cases includesilicon and hydrocarbon oil/surfactant systems.

As alluded to above, the devices described herein are useful inproviding the microcapsules within aqueous droplets in an immisciblefluid. As will be appreciated, in a number of applications, it isparticularly beneficial to provide a desired level of microcapsuleoccupancy in created partitions. In general, this is accomplished bycontrolling the combination of the aqueous stream that includes themicrocapsule, and the streams of the immiscible fluid, such that theprobability of more than the desired number of microcapsules beingincorporated into a given partition is acceptably low. This maygenerally be accomplished through control of the flow of microcapsules,along with the flow of the other fluids coming together in thepartitioning zone, e.g., junction 322 in FIG. 3, can be controlled so asto substantially provide for a desired number of microcapsules perpartition.

In many cases, the devices, systems and methods are used to ensure thatthe substantial majority of occupied partitions (e.g., partitionscontaining one or more microcapsules) will include no more than 1microcapsule per occupied partition. In particular, in some cases, thepartitioning process is controlled such that fewer than 50% of theoccupied partitions contain more than one microcapsule, fewer than 45%of the occupied partitions contain more than one microcapsule, fewerthan 40% of the occupied partitions contain more than one microcapsule,fewer than 35% of the occupied partitions contain more than onemicrocapsule, fewer than 30% of the occupied partitions contain morethan one microcapsule, fewer than 25% of the occupied partitions containmore than one microcapsule, and in many cases, fewer than 20% of theoccupied partitions have more than one microcapsule, while in somecases, fewer than 10% or even fewer than 5% of the occupied partitionswill include more than one microcapsule per partition. Accordingly, inmany cases, the resulting partitions will result in at least 50% of thepartitions containing one and only one microcapsule (i.e., a singlemicrocapsule), at least 55% of the partitions containing one and onlyone microcapsule, at least 60% of the partitions containing one and onlyone microcapsule, at least 65% of the partitions containing one and onlyone microcapsule, at least 70% of the partitions containing one and onlyone microcapsule, at least 75% of the partitions containing one and onlyone microcapsule, at least 80% of the partitions containing one and onlyone microcapsule, at least 80% of the partitions containing one and onlyone microcapsule, at least 85% of the partitions containing one and onlyone microcapsule at least 90% of the partitions containing one and onlyone microcapsule, and in some cases at least 95% of the partitionscontaining one and only one microcapsule.

Additionally or alternatively, in many cases, it is desirable to avoidthe creation of excessive numbers of empty partitions. While this may beaccomplished by providing sufficient numbers of microcapsules into thepartitioning zone, the poissonian distribution can expectedly increasethe number of partitions that can include multiple microcapsules. Assuch, in accordance with aspects of the present disclosure, the flow ofone or more of the microcapsules, or other fluids directed into thepartitioning zone are controlled such that, in many cases, no more than50% of the generated partitions will be unoccupied, i.e., including lessthan 1 microcapsule, no more than 25% of the generated partitions, or nomore than 10% of the generated partitions, will be unoccupied. Further,in some aspects, these flows are controlled so as to presentnon-poissonian distribution of single occupied partitions whileproviding lower levels of unoccupied partitions. Restated, in someaspects, the above noted ranges of unoccupied partitions will beachieved while still providing any of the above-described singleoccupancy rates described above. For example, in many cases, the use ofthe devices, systems and methods of the present disclosure createsresulting partitions that have multiple occupancy rates of from lessthan 25%, less than 20%, less than 15%, less than 10%, and in manycases, less than 5%, while having unoccupied partitions of from lessthan 50%, less than 40%, less than 30%, less than 20%, less than 10%,and in some cases, less than 5%. Methods, systems and deviceconfigurations for controlling the various flows within the channelnetworks are described in greater detail below.

Although described in terms of providing substantially singly occupiedpartitions, above, in certain cases, it is desirable to provide multiplyoccupied partitions, e.g., containing two, three, four or moremicrocapsules within a single partition. Accordingly, as noted above,the flow characteristics of the microcapsule containing fluids andpartitioning fluids may be controlled to provide for such multiplyoccupied partitions. In particular, the flow parameters may becontrolled to provide a desired occupancy rate at greater than 50% ofthe partitions, greater than 75%, and in some case greater than 80%,90%, 95%, or higher.

Additionally, in many cases, the multiple microcapsules within a singlepartition may comprise different reagents encapsulated therein. In suchcases, it may be advantageous to introduce different microcapsules intoa common channel or droplet generation junction, from differentmicrocapsule sources, i.e., containing different encapsulated reagents,through different channel inlets into such common channel or dropletgeneration junction. In such cases, the flow and frequency of thedifferent microcapsules into the channel or junction may be controlledto provide for the desired ratio of microcapsules from each source,while ensuring the desired pairing or combination of such microcapsulesinto a partition.

Although shown with two junctions and their associated channel segments,it will be understood that additional channels may be provided withinthe devices of the present disclosure to deliver additional componentsto the various fluids, capsules and partitions described above. Theseadditional channels may be provided intersecting any of the variouschannel segments described herein for addition of a variety ofcomponents to any one or more of the various fluids flowing within thosechannel segments at different positions and for different purposes. Forexample, in one aspect, one or more additional side channels may beprovided intersecting the channel segment 328, described above, for thepurpose of introducing new fluids, reagents, or additional partitioningfluids into partitioned fluids within the channel segment 328.

Likewise, additional channel segments may be provided intersectingchannel segments 302 and/or 308, in order to introduce additional fluidsinto the aqueous stream prior to separating that fluid stream intodroplets with the partitioning fluid. Additionally, still other channelsegments can be provided intersecting any of the side channel segments,e.g., channel segments 304, 306, 324, or 326, in order to deliverdifferent fluids into those channels. Such systems can allow thealteration of fluids being introduced into the partitioning stream inreal time by controlling which fluids are provided through therespective side channels, e.g., allowing one to change reactants, changethe partition fluid characteristics, or any of a variety of otherconditions.

In some cases, these additional fluids may be for purposes ofstimulating different reactions within the partitions by introducing newreagents to the partitions. For example, these additional fluids mayprovide one or more activating agents to the partitions or capsules,that cause the initiation of one or more reactions at any stage prior toor following partitioning.

Such activating agents may take any of a number different forms. Forexample, these activation reagents may cause the release of a reagentwithin a partition or capsule, to make it available for reaction, e.g.,by cleaving a linkage between a microcapsule and the reagent, or bystimulating the disintegration of the microcapsule and subsequentreagent release. Alternatively or additionally, the activation reagentmay comprise an initiator for a desired reaction, such as a missingcritical reagent for the desired reaction, or the like. By way ofexample and for purposes of illustration, in cases where the desiredreaction includes a nucleic acid polymerase mediated nucleic acidreplication, an activation reagent may include a key missing reagent,such as one or more nucleoside triphosphates otherwise lacking from themixture, a primer sequence, or one or more reaction co-factors suitablefor a polymerase reaction, e.g., divalent metal ions like magnesium ormanganese. In many cases, the use of such missing systems or activatablereagent systems for purposes of controlled initiation of a givenreaction are referred to as “hot start” reagents, which are, as ageneral class, useful in conjunction with the systems of the presentdisclosure.

The activation reagents may alternatively or additionally initiatereactions on the partitions or capsules themselves or both, for example,disrupting the capsules or releasing reagents from those capsules,stabilizing or destabilizing partitions, e.g., to reduce or promotecoalescence, respectively. A variety of reagent systems may be employedin the disruption of or release of reagents from the microcapsules ofthe present disclosure. These include the use of chemical stimulidescribed above, for cleaving chemical cross-linking or molecularattachment, as discussed in U.S. Patent Publication No. 2014/0378345,which is entirely incorporated herein by reference.

FIG. 4 provides a schematic illustration of an overall exemplarymicrofluidic device or device module for partitioning encapsulatedreagents as described above. As shown in FIG. 4, the overall device 400provides one or more channel network modules 450 for generatingpartitioned microcapsule compositions. As shown, the channel networkmodule 450 includes a basic architecture similar to that shown in FIG.3B, above. In particular, the illustrated channel network moduleincludes a first channel junction 410 linking channel segments 402, 404and 406, as well as channel segment 408 that links first junction 410 tosecond channel junction 422. Also linked to second junction 422 arechannel segments 424, 426 and 428.

As illustrated, channel segment 402 is also fluidly coupled to reservoir430 that provides, for example, a source of microcapsules that mayinclude one or more encapsulated reagents, suspended in an aqueoussolution. Each of channel segments 404 and 406 are similarly fluidlycoupled to fluid reservoir 432, which may provide for example, a sourceof sample material as well as other reagents to be partitioned alongwith the microcapsules. As noted previously, although illustrated asboth channel segments 404 and 406 being coupled to the same reservoir432, these channel segments may be coupled to different reservoirs forintroducing different reagents or materials to be partitioned along withthe microcapsules.

Each of channel segments 402, 404 and 406 may be provided withadditional fluid control structures, such as passive fluid valve 436.These valves may provide for controlled filling of the overall devicesby breaking the capillary forces that draw the aqueous fluids into thedevice at the point of widening of the channel segment in the valvestructure. Briefly, aqueous fluids are introduced first into the devicein reservoirs 430 and 432, at which point these fluids will be drawn bycapillary action into their respective channel segments. Upon reachingthe valve structure, the widened channel will break the capillaryforces, and fluid flow will stop until acted upon by outside forces,e.g., positive or negative pressures, driving the fluid into and throughthe valve structure. Although illustrated as a widening of the channelin the width dimension, it will be appreciated that a passive valvestructure may include a step up in any one or more cross-sectionaldimensions of a channel region. For example, a passive valve mayincrease an increased stepped depth of a channel at the valve region.Again, when the fluid reaches the increased cross sectioned channelsegment, the capillary forces will retain the fluid within the shallowerchannel. Again, as noted, the increase in cross-sectional dimension canbe in any one or more cross-sectional dimensions, and may be increasesin cross section of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% 100%, or even more. In many cases, it may be between about5% and about 100% larger cross section, between about 5% and about 50%,between about 5% and about 20% of an increase in cross section. Althoughillustrated at a particular channel location, it will also beappreciated that these valve structures may be positioned along anychannel location within a microfluidic channel network, including at anintersection of two or more channel segments, or within a singularchannel.

Also shown in channel segment 402 is a microcapsule funneling structure452, that both allows the efficient gathering of microcapsules fromreservoir 430, regulation of microcapsule flow (as described in greaterdetail elsewhere herein), as well as reduced system failure due tochannel clogging. As also shown, in some cases, the connection ofchannel segment 402 with reservoir 430, as well as the junctions of oneor more or all of the channel segments and their respective reservoirs,may be provided with additional functional elements, such as filteringstructures 454, e.g., pillars, posts, tortuous fluid paths, or otherobstructive structures to prevent unwanted particulate matter fromentering or proceeding through the channel segments.

Junction 410 is fluidly coupled to second junction 422. Also coupled tochannel junction 422 are channel segments 424 and 426 that are, in turnfluidly coupled to reservoir 438, which may provide, for example,partitioning fluid that is immiscible with the aqueous fluids flowingfrom junction 410. Again, channel segments 424 and 426 are illustratedas being coupled to the same reservoir 438, although they may be coupledto different reservoirs, e.g., where each channel segment is desired todeliver a different composition to junction 422, e.g., partitioningfluids having different make up, including differing reagents, or thelike.

In exemplary operation, microcapsules provided in reservoir 430 areflowed through channel segment 402 into first channel junction 410. Themicrocapsules will flow through valve 436, which, in addition toproviding a passive fluid valve structure also operates as amicrocapsule flow regulator, as described in greater detail below. Themicrocapsule flow regulator ensures more regular flow of microcapsulesinto and through junction 410 into channel segment 408. Within junction410, the aqueous microcapsule solution is contacted with the aqueousfluids from reservoir 432, as introduced by channel segments 404 and406. Due to laminar flow characteristics of the microfluidic channelnetworks, and without being bound to any particular theory of operation,aqueous fluids from channel segments 404 and 406 can ensheath themicrocapsule composition with a second aqueous fluid layer, where theprimary interaction between the two fluids is through simple diffusion,i.e., with a substantial lack of convective mixing.

The aqueous fluid stream is then flowed through channel segment 408 intosecond junction 422. Within channel junction 422, the aqueous fluidstream, including the regularly spaced flowing microcapsules, flowingthrough channel segment 408, is formed into droplets within theimmiscible partitioning fluid introduced from channel segments 424 and426. In some cases, one or both of the partitioning junction, e.g.,junction 422 and one or more of the channel segments coupled to thatjunction, e.g., channel segments 408, 424, 426 and 428, may be furtherconfigured to optimize the partitioning process at the junction.

Further, although illustrated as a cross channel intersection at whichaqueous fluids are flowed through channel segment 408 into thepartitioning junction 422 to be partitioned by the immiscible fluidsfrom channel segments 424 and 426, and flowed into channel segment 428,as described elsewhere herein, partitioning structure within amicrofluidic device of the present disclosure may comprise a number ofdifferent structures.

As described in greater detail elsewhere herein, the flow of themicrocapsules into junction 422, and in some cases the rate of flow ofthe other aqueous fluids and/or partitioning fluid through each ofjunctions 410 and 422, are controlled to provide for a desired level ofpartitioning of microcapsules, e.g., to control the number ofmicrocapsules that will be partitioned in each droplet, the amount ofreagents in each droplet, and control the overall operation of thedevice, e.g., to prevent clogging or other disruption, or the like.

Once the microcapsules are partitioned, they are flowed through channelsegment 428 and into a recovery structure or zone where they may bereadily harvested. As shown, the recovery zone includes, e.g., outletreservoir 438. Alternatively, the recovery zone may include any of anumber of different interfaces, including fluidic interfaces with tubes,wells, additional fluidic networks, or the like. In some cases, wherethe recovery zone comprises an outlet reservoir, the outlet reservoirwill be structured to have a volume that is greater than the expectedvolume of fluids flowing into that reservoir. In its simplest sense, theoutlet reservoir may, in some cases, have a volume capacity that isequal to or greater than the combined volume of the input reservoirs forthe system, e.g., reservoirs 430, 432 and 434.

As will be appreciated, a single microfluidic device may includemultiple substantially identical channel network modules that may eachhave self-contained fluid sources or may share one or more fluidreservoirs. For example, a single multiplexed device including multiplechannel network modules may include a single source of one or more ofthe partitioning fluid, the microcapsule containing fluid, one or morereagent fluids, as well as sample fluids. As such, the multiple channelmodules can be used to generate large amounts of the same type ofpartitioned microcapsules, e.g., by providing the same allocation offluids in the corresponding reservoirs of each module 450 in amultiplexed device. In certain aspects, however, different channelnetwork modules will be used in the generation of different partitionedmicrocapsules. Such different partitioned compositions may includedifferent sample materials being allocated to the partitionedmicrocapsules, different initial microcapsules being allocated to thesame or different sample materials, or application of different reagentsto different to the same or different sample materials and/or differentmicrocapsules. As noted above, where the same fluids are beingintroduced into the channel segments of different modules, it can beefficient to have such channel segments fluidly coupled to the samereservoir(s). These channel segments may be the same correspondingchannel segments in each module or, depending upon the desired use, theymay be different channel segments in different modules.

As will be appreciated, the rates at which different fluids are broughttogether in the channel structures described above can have an impact onthe generation of the droplets whether for the purpose of microcapsulegeneration or for their subsequent separation into discrete partitionsor droplets. Accordingly, in certain aspects, the devices used in thepresent disclosure provide for control of the various fluid flows withinthe integrated channel networks. Control of fluid flows within channelnetworks may be accomplished through a variety of mechanisms. Forexample, pressures may be applied at the origin of different channelsegments, e.g., on reservoirs, in order to control fluid flow withinthat channel segment. By utilizing a pressure based flow, one may beable to independently control flows within different channel segments bycoupling independently controlled pressure sources to the differentchannel segments to apply differential pressure gradients across eachchannel segment. In such cases, flow rates within different channelsegments may be monitored, e.g., through interfaced detection systems,such as optical detectors, to provide feedback on the flow controlaspects to allow modulation of flow.

Alternatively, a single pressure source may be coupled to all channelsegments simultaneously, e.g., by coupling a pressure source to amanifold that simultaneously connects to the various channel segmentorigins or reservoirs. Where a single pressure is applied over multiplechannels, the flow rates within those channels will be controlled by thelevel of resistance within each channel that is subject to fluidviscosity and channel dimensions (cross-section and length). In suchcases, flow control is achieved by providing channel segments with theappropriate dimensions to achieve the desired flow rate given theviscosity of the fluids passing through it. By way of example, in orderto achieve equivalent flow rates, channels used to flow more viscousfluids may be provided with wider and/or shorter channel segments thanchannels used to transport lower viscosity fluids.

Although described as a pressure source applied to channel origins, insome aspects, the pressure source may include a vacuum (or negativepressure) source that is applied to one or more of the outlet ports fora channel network, e.g., a terminal reservoir, i.e., reservoir 444 inFIG. 4. Application of a vacuum provides a number of advantages overpositive pressure driven systems, including, e.g., provision of a singlepoint of connection to an integrated channel network at the outlet vs.several inlet points, lack of microcapsule compression that may lead tochannel inlet clogging in positive pressure systems, and the like.

In some cases, for the partitioning of microcapsules, the vacuum sourcemay be applied to a node on an outlet channel segment that is distinctfrom the zone at which the partitioned microcapsules may be harvested.In particular, where a vacuum source is applied at the terminalreservoir, e.g., reservoir 438 in FIG. 4, the source can be disconnectedfrom the reservoir in order to harvest the partitioned microcapsulesfrom the terminal reservoir. In some cases, by separating the vacuumsource interface node with the channel segment from the zone wherepartitioned microcapsules are harvested, one can obviate the need fordisconnecting the vacuum source and improving the ease of use. In somecases, the vacuum interface node may include a terminal reservoir, e.g.,reservoir 438, which may be configured with an interface component forinterfacing with an integrated or discrete partition harvesting zonethat allows harvesting of the partitions without removing the connectedvacuum source. These and other interface components are described indetail below.

III. Additional Improved Microfluidic System Components

The precise handling and manipulation of microcapsules, either in theircreation, or in their subsequent partitioning, creates a number of newchallenges in microfluidic systems that are addressed by aspects of thepresent disclosure. In particular, flow of microcapsule in fluidic andespecially microfluidic systems can be subject to certain variabilitiesmany of which have been alluded to above, including varied flow rates ordispensing frequencies, channel clogging, variable partitioning,sampling or dispensing biases, or the like. This disclosure providesnumerous improved components, devices, methods and systems foraddressing many of these issues.

For example, in certain aspects, the present disclosure addresses, e.g.,sampling biases or variability from microcapsules in a reservoir. Inparticular, in some cases, one or more reservoirs into whichmicrocapsules are deposited in a system or device described herein,e.g., reservoir 430 shown in FIG. 4, are configured to improve the flowof microcapsules into their connected channel segments.

In one example, the reservoirs that are used to provide themicrocapsules or other reagents may be provided with a conical bottomsurface to allow for funneling of the microcapsules toward the inletsfor the channel segments connected to the reservoirs. This isschematically illustrated in FIG. 5A, which shows an example ofreservoirs 500, 502, 504 and 506, viewed from the side. As shown, thereservoir 500 includes side walls 510 that extend from an upper surface512 of a microfluidic device 506. An interior cavity portion 508 of thereservoir extends into the microfluidic device 506 and is provided incommunication with a fluidic channel 516. As shown, cavity portion 508possesses a tapering or conical shape toward the inlet of channel 516,as defined by narrowing of the cavity 508, by virtue of convergingsidewalls 518 of cavity 508.

In additional aspects, microcapsule loading into channel segments may beenhanced through the inclusion of a broadened interface region, orinlet, between the reservoir and the connected fluid channel. Oneexample of this is illustrated in the channel network of FIG. 4, wherethe interface of channel segment 402 with reservoir 430 is provided withfunneling channel structure 452, that both enhances the introduction ofmicrocapsules into the channel segment, as well as provides some flowregulating characteristics for the microcapsules into the channelsegment. Also shown, are obstructive structures 454, that providebarriers for larger particulate matter that may be a contaminant withinthe reservoir and may impair the flow of fluids through the channels ofthe device. As will be appreciated, the various reservoirs may each orall include filtration or particle blocking elements within them thatmat be the same or different, depending upon the fluids to be disposedin the reservoir. For example, in some cases, while a simple structuralbarrier, like the pillar structures shown in FIG. 4 (e.g., structures454) may be used in the channel interfaces with the microcapsulecontaining reservoirs, for those reservoirs containing aqueoussolutions, e.g., sample materials or reagents, more or less stringentfiltration components may be integrated therein, e.g., at the bottom ofa reservoir, in order to filter the contents of the reservoir, in situ,to a greater or lesser degree. A variety of filtration media, including,e.g., membrane filters, frits, or other known filter types, can bereadily incorporated into the reservoirs within the devices of thepresent disclosure.

Similar to the broadened interfaces described above, the interfaces mayinclude multiple discrete channel inlets from a given reservoir, toensure that the flow of microcapsules into and through the channelsegments is less susceptible to interruption or clogging, as well as toensure that microcapsules disposed in the reservoir are accessed atmultiple points, rather than at a single point or channel inlet. Inparticular, for a given reservoir, there may be provided a plurality ofchannel inlets that fluidly connect the reservoir to a single channelsegment (or flow regulating junction, as described in greater detailbelow) within the microfluidic device. Further, as described above, themultiple channel inlets may be provided with one or more of thefunctional elements described previously, e.g., funneling structures,filtering elements such as pillars, posts or tortuous paths, or thelike.

As noted in the discussion of the microcapsule partitioning above, theflow of microcapsules, along with the flow of the other fluids comingtogether in the partitioning zone, e.g., junction 322 in FIG. 3, can becontrolled so as to substantially provide for a desired number ofmicrocapsules per partition. In many cases, the substantial majority ofoccupied partitions (e.g., partitions containing one or moremicrocapsules) will include no more than 1 microcapsule per occupiedpartition, while in some cases also reducing the number of unoccupiedpartitions created.

As described above, the methods, devices and systems of the presentdisclosure generally accomplish a desired level of allocation ofmicrocapsules to partitions through the controlled combination of themicrocapsules and partitioning or dispersion fluid into droplets, e.g.,through controlling the flow rates of microcapsules and oil in to thedroplet generating junction of a microfluidic device, i.e., junction 312as shown in FIG. 3.

Flowing of microcapsules from reservoirs through channels and intochannel junctions can be subject to a great deal of variability, asthese microcapsules may flow at a that is defined by the happenstance ofwhen the microcapsule enters a channel segment, and its flow ratethrough that channel segment. Accordingly, in certain aspects, themicrofluidic systems of the present disclosure may include microcapsuleflow regulator components within the appropriate channel segment toprovide such microcapsules flowing into the droplet forming region at amore defined regularity.

The microcapsule flow regulators included within the channel systemsdescribed herein will typically provide microcapsules flowing withinchannels at a relatively regular frequency. In particular, during agiven timeframe in which droplets are being generated, e.g., a 10 secondwindow, a 30 second window, a one minute window, a 2 minute window, a 3minute window, or over the steady state operation of an entire dropletgeneration run (e.g., not including start up and shut down), thefrequency at which these microcapsules are flowing will typically have acoefficient of variation of less than 50%, less than 40%, less than 30%,less than 20%, less than 10%, and in some cases, less than 5%. As willbe appreciated, the flow frequency of microcapsules reflects the numberof microcapsules that flow past a given point in a conduit within a onesecond period of time. Frequency measurements may typically be basedupon sub-second or one second intervals, but may also be based uponmultiple second, multiple minute or longer intervals, depending upon theparticular needs of the process.

Although in a given process, it may be desirable to flow microcapsulesat a relatively stable frequency, in a number of aspects, the frequencyfor the flowing microcapsules can differ depending upon the desiredapplications, the nature of the fluids being flowed, and the like. Ingeneral, however, microcapsules being flowed into a partitioning ordroplet generating junction are flowed at greater than 50 Hz, greaterthan 100 Hz, greater than 250 Hz, greater than 500 Hz, greater than 750Hz, greater than 1000 Hz, greater than 1500 Hz, greater than 2000 Hz, oreven greater than 5000 Hz or even 10,000 Hz, while still achieving thedesired occupancy and other process goals. In certain cases, these flowfrequencies may be maintained after the partitioning junction, such thatpartitioned microcapsules are flowing out of the droplet generationjunction at frequencies of at least or greater than 50 Hz, at least orgreater than 100 Hz, at least or greater than 500 Hz, at least orgreater than 750 Hz, at least or greater than 1000 Hz, at least orgreater than 1500 Hz, at least or greater than 2000 Hz, or even at leastor greater than 5000 Hz or 10,000 Hz.

A number of approaches may be adopted to regulate bead flows within themicrofluidic channel segments of the devices described herein. Forexample, in some cases, these regulators include “gathering zones” inwhich the microcapsules will flow into and gather before flowing out ofthe gathering zone. These zones are configured to more effectively meterthe flow of the microcapsules through the inclusion of funnelingstructures or channel profiles to better meter the flow of individualmicrocapsules. Examples of such structures are alluded to above, and areshown in FIGS. 4 and 6B. A first example includes the channel interfaceshown as funneling zone 452 integrated within the interface of channelsegment 402 and reservoir 430.

In a similar fashion, a microcapsule flow regulator may be integratedwithin the channel segment itself, e.g., channel segment 402 in FIG. 4,rather than at the interface with the reservoir, e.g., reservoir 430 ofFIG. 4. An example of this structure includes the flow regulatorstructure 600 illustrated in FIG. 6A. As will be appreciated, themicrocapsule flow regulating structure 460 may also function as apassive fluid valve during filling of the device, e.g., valve 436, asdescribed with respect to channel segments 402, 404 and 406, above. Aswith funneling structure 452, flow regulator 600 includes a broadenedregion of channel segment 602 (shown at the interface as channel 602 a)at region 604 that narrows at region 606 until it rejoins thecross-sectional dimensions of the outlet portion of channel segment 602(shown at the interface as segment 602 b). As the microcapsules enteredthe expanded region 602, the convective flow will allow multiplemicrocapsules to gather or aggregate within the overall gatheringregion. Once sufficient numbers of microcapsules have aggregated, theywill begin to flow out through the narrowed region into channel segment604 in a metered and more controlled manner. This is schematicallyillustrated in FIG. 6B, showing microcapsules flowing at irregularfrequency into the microcapsule flow regulating structure, and flowingout of the regulator at a more regular frequency. As will beappreciated, a channel network may include one or more flow regulatorsarranged in series or in parallel within a given flow path, e.g., thefluid connection between two points in the overall network. While theseflow regulators may include those having the shape and configuration asshown in FIGS. 6 A and 6B, they may also include different shapes andconfigurations. For example, the broadened regions of the flow regulatormay include triangular shapes similar to that shown in FIGS. 6A and 6B,or may include elongated triangular shapes. Likewise, the broadenedregion of the flow regulators may include circular, elliptical orsemi-circular or semi-elliptical shapes, or may include a tapered funnelshape like the channel interfaces described elsewhere herein. As will beappreciated, the basic structural components of these exemplary flowregulators is a broadened channel region at the point a flow enters intothe regulator, with a tapered, narrowing or funneling portion as theflow enters into the subsequent channel or channel network. Thesebroadened regions will typically have wider cross sections that are from1.1× to 20× the cross section of channel segments flowing into thebroadened region. In some aspects, these broadened regions are anywherefrom 2× to 10× the cross-section of the entering channel segment (ascompared against the same cross-sectional measurement, e.g., width towidth, depth to depth, etc.), and in some cases, from 2× to 5× the crosssection of the entering channel segment. In some cases, more than onecross-sectional dimension may be varied over the inlet channel, e.g.,both width and depth may be different. Further, although in someaspects, where both dimensions are varied, they will be greater thanthose of the inlet channel, in some cases, provided at least one ofwidth and depth is increased, the other dimension may be decreased,depending upon the desired flow characteristics through the flowregulator.

In other examples, multiple microcapsule containing channels are broughttogether at a gathering zone to bring in a higher number ofmicrocapsules into the junction and its connected effluent channelsegment. This allows voids in the flow of microcapsules in one channelto be filled by microcapsules flowing in from the other channel(s).These channel segments may include separate channel segments providedwithin the channel network as a gathering zone, or as noted above, theymay comprise multiple inlet channel segments that are fluidly connectedto a microcapsule containing reservoir. Further, as noted previously,these channel segments may deliver microcapsules from a single source orpopulation of microcapsules to the same channel segment, or they maydeliver microcapsules from different sources, e.g., reservoirs, to acommon channel segment, where such different microcapsules includedifferent reagents.

As noted above, the microfluidic devices and systems of the presentdisclosure may include improved interface components useful in operationof the devices and systems, and interface components that may beparticularly useful in the handling and manipulation of microcapsulecompositions and partitioned compositions.

Examples of interfaces useful for microcapsule and partitionmanipulation include those useful for one or both of deposition andharvesting of such compositions to and from such devices. For example,as noted previously, movement and transport of microcapsules in solutioncan be subject to some variability. This variability can, in someinstances, carry over to transport of these solutions from the systemsin which they are created into other systems and/or vessels, e.g.,storage vessels such as tubes, wells, vials, or the like, or intransporting them from storage vessels, e.g., tubes, wells, vials or thelike, into systems for their subsequent processing, e.g., microfluidicpartitioning systems like those described above. In one example, amicrocapsule solution or suspension is provided within a storage vesselthat includes a pierceable wall or base surface. Corresponding piercingstructures may be provided within a reservoir on a fluidic device. Byinserting the storage vessel into the reservoir, the pierceable wall ispenetrated by the piercing structures to release the microcapsulesuspension into the reservoir.

An example of this type of interface is schematically illustrated inFIGS. 7A and 7B. As shown in FIG. 7A, a storage vessel, such as tube 702is provided for holding fluid reagents, such as a microcapsulesuspension 704, as described elsewhere herein. A surface of the vessel,e.g., base surface 706 is provided as a pierceable layer. Pierceablelayers may be provided in any of a variety of different configurations.For example, they may simply include walls of the same material as therest of the vessel, but which are sufficiently thin to allow piercing.Such walls may be thinner than other walls in the vessel. Alternatively,the pierceable surfaces may include different materials from that of theremainder of the vessel, such as a pierceable septum (e.g.,nitrocellulose, PVDF, latex, or any other similarly used materials), afoil surface, or any of a number of other pierceable membranes.Likewise, a surface of the storage vessel may be provided with a valvingstructure that may be active or passive. In many cases, passive valves,such as pressure triggered check valves may be employed in base surface706 of the storage vessel.

In use, the storage vessel is mated with the reservoir 708 in a device710, as shown in FIG. 7B. Reservoir 708 is configured with piercingstructures 712 that are positioned to contact and penetrate the basesurface 706 of the storage vessel when the vessel is inserted into thereservoir 708. Once inserted, the base surface 706 is ruptured and themicrocapsule suspension 704 contained in vessel 702 is permitted todrain into reservoir 708. In some cases, vessel 702 may be provided withadditional components to facilitate driving of the suspension into thereservoir, such as a plunger or other pressurizing device, to force thesuspension from the vessel. In other cases, simple gravity flow may beused to transfer the suspension. In some cases, the piercing structureand wall or base component of the vessel maybe configured to optimizethe transfer of the suspension from the vessel to the reservoir, throughthe inclusion of hydrophobic interior coatings on the vessel,flash-mitigating piercing structures (e.g., to reduce the possibilitythat remnants of the pierced surface may block flow of the suspensionout of the vessel). In alternate aspects, dissolvable, degradable orotherwise activatable barriers may be provided in order to allow for thecontrolled dispensing of the suspension. Such barriers include, e.g.,dissolvable films or membranes that are degraded, dissolved or renderedsufficiently permeable to dispense the suspension upon application of astimulus. Such barriers may be degraded upon application of a specificchemical, thermal, electromagnetic, or other stimulus.

Similar to the interfaces described above, in some cases for harvestingeither microcapsules or partitioned microcapsules or other materialsfrom devices, such interface components may include, e.g., a pierceablebase layer for the harvesting reservoir, e.g., reservoir 216 shown inFIG. 2, or reservoir 438 of the device illustrated in FIG. 4, to allowaccess to and removal of partitioned microcapsules from the terminalreservoir without necessarily removing the interfaced vacuum source. Inoperation, at the conclusion of a partitioning operation, the base ofthe terminal reservoir may be pierced, and the generated partitions areeither removed or allowed to drain or flow into a waiting receptacle,e.g., by reversing the vacuum source to apply pressure to the reservoir438, to drive the partitioned microcapsules through the pierced baselayer of the reservoir, or through gravity driven flow. This waitingreceptacle may be integrated into the device, or positioned adjacent tothe microfluidic device in order to receive the partitionedmicrocapsules.

In other examples, one or both of the reservoir and storage vessel maybe configured to provide efficient transfer from one to the other. Forexample, in some cases, a vessel including a microcapsule suspension maybe provided with an interface component that allows it to be mated,connected and/or coupled directly to the receiving reservoir toefficiently transfer its contents. In some cases, the connection may bebounded by a check valve to prevent movement of the suspension until anappropriate driving force is applied to the suspension.

In addition to fluidic interfaces, the devices and systems describedherein may also include one or more of a variety of mechanical orphysical interface components. Such components include, for example,handling components to facilitate the manual or automated movement andhandling of the devices, alignment components, to ensure properplacement and alignment of the devices on instruments, holders and thelike, as well as functional components, to allow for additionalmanipulation of sample materials within the devices. Examples ofhandling components include tabs, walls, or other surfaces that arepositioned away from critical or sensitive surfaces of a device (e.g.,optical windows, surfaces prone to contamination, etc.), as well assurfaces that are configured to facilitate handling, whether manual orautomated, e.g., with sufficient size and/or textured surfaces to ensuregrip and control.

Examples of alignment structures include mechanical elements that ensurealignment of a device with a corresponding instrument, or other fixture,such as beveled corners, device shapes, and integrated key elements(e.g., tabs, slots, posts, or the like) that mate with complementarystructures on the other fixture. Such alignment components also includeoptically detected components, such as registration marks or fiducials,barcode tags, or other machine readable components integrated into orattached to a device.

A wide variety of functional components or functional componentinterfaces are also envisioned, including, e.g., those interfacecomponents that are important for operation of the device. Examples ofsuch interface components include, for example, gasket structures thatmay be integrated into or separately placed over the upper surfaces ofone or more reservoirs, to ensure sealed application of pressures orvacuums to the devices described herein. In certain aspects, thesegaskets will be either integrated into the device, or provided as aseparate, disposable component, rather than being integrated into aninstrument, in order to minimize the possibility of instrumentcontamination. Other examples of functional interface components includeinterfaces for mixing or agitating components within the reservoirs.Such components are useful in come cases to prevent settling ofmicrocapsule compositions. These interfaces may comprise actualagitation components, such as piezoelectric, acoustic, or mechanicalvibration components integrated into the devices, or they may comprisesurfaces that are suitable for or are configured to interface thesecomponents on a corresponding instrument system or other fixture.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. For example, particle delivery canbe practiced with array well sizing methods as described. Allpublications, patents, patent applications, and/or other documents citedin this application are incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, patent application, and/or other document were individually andseparately indicated to be incorporated by reference for all purposes.

1. A method for partitioning microcapsules, comprising: (a) providing anaqueous fluid comprising a suspension of microcapsules; and (b) flowingthe aqueous fluid into a droplet generation junction comprising apartitioning fluid to form a population of droplets of the aqueous fluidin the partitioning fluid, wherein a flow rate of the aqueous fluid issuch that no more than 50% of droplets of the population of droplets areunoccupied by a microcapsule from the suspension of microcapsules. 2.The method of claim 1, wherein the flow rate is such that no more than25% of the droplets of the population of droplets are unoccupied by amicrocapsule.
 3. The method of claim 2, wherein the flow rate is suchthat no more than 10% of the droplets of the population of droplets areunoccupied by a microcapsule.
 4. The method of claim 1, wherein fewerthan 25% of droplets of the population of droplets comprise more thanone microcapsule.
 5. The method of claim 4, wherein fewer than 20% ofdroplets of the population of droplets comprise more than onemicrocapsule.
 6. The method of claim 5, wherein fewer than 15% ofdroplets of the population of droplets comprise more than onemicrocapsule.
 7. The method of claim 6, wherein fewer than 10% ofdroplets of the population of droplets comprise more than onemicrocapsule.
 8. The method of claim 7, wherein fewer than 5% ofdroplets of the population of droplets comprise more than onemicrocapsule.
 9. The method of claim 1, wherein at least 80% of dropletsof the population of droplets comprise a single microcapsule.
 10. Themethod of claim 9, wherein at least 90% of droplets of the population ofdroplets comprise a single microcapsule.
 11. The method of claim 10,wherein at least 95% of droplets of the population of droplets comprisea single microcapsule.
 12. The method of claim 1, wherein the dropletgeneration junction is in a microfluidic channel network of amicrofluidic device.
 13. The method of claim 12, wherein themicrofluidic channel network comprises a first channel segment fluidlyconnecting a source of microcapsules to the droplet generation junction,a second channel segment connecting a source of partitioning fluid tothe droplet generation junction, and a third channel segment fluidlyconnected to the droplet generation junction providing an outlet to thedroplet generation junction.
 14. The method of claim 13, wherein (b)comprises providing one or more pressure differentials across the firstand second channel segments to provide the flow rate.
 15. The method ofclaim 13, wherein the first and/or second channel segments havecross-sectional dimensions that provide the flow rate such that no morethan 50% of droplets of the population of droplets are unoccupied by amicrocapsule from the suspension of microcapsules.
 16. The method ofclaim 13, wherein the microfluidic channel network further comprises oneor more flow controlling structures within the first channel segmentthat provide the flow rate.
 17. A method for partitioning microcapsules,comprising (a) flowing an aqueous fluid comprising a suspension ofmicrocapsules into a droplet generation junction comprising apartitioning fluid, wherein during a window of droplet generation, themicrocapsules are flowing into the droplet generation junction at afrequency that varies less than 30%; and (b) partitioning themicrocapsules in the partitioning fluid during the window of dropletgeneration. 18.-24. (canceled)
 25. A method for producing microcapsules,comprising: (a) providing a gel precursor in an aqueous fluid; (b)flowing the aqueous fluid having the gel precursor through a fluidconduit that is fluidly connected to a droplet generation junctioncomprising a partitioning fluid, wherein the partitioning fluidcomprises a gel activation agent; and (c) forming droplets of theaqueous fluid in the partitioning fluid, wherein within the droplets,the gel activation agent contacts the gel precursor to form gelmicrocapsules.
 26. (canceled)
 27. A method for partitioningmicrocapsules, comprising: (a) flowing an aqueous fluid comprising asuspension of a monodisperse population of microcapsules into a dropletgeneration junction, the monodisperse population having a meancross-sectional dimension and a coefficient of variation incross-sectional dimension of no greater than 10%; (b) introducing apartitioning fluid into the droplet generation junction; and (c)separating the aqueous fluid into droplets within the partitioningfluid, the droplets containing one or more microcapsules.
 28. Amicrofluidic system, comprising: (a) a microfluidic channel networkcomprising at least first, second and third channel segments in fluidcommunication with a droplet generation junction; (b) a first fluidsource fluidly connected to the first channel segment and comprising afirst fluid that comprises an aqueous fluid, the aqueous fluidcomprising a plurality of microcapsules disposed therein; (c) a secondfluid source fluidly connected to the second channel segment andcomprising a second fluid that is immiscible with the aqueous fluid; and(d) a flow control system connected to the microfluidic channel networkthat (i) subjects the first fluid and second fluid to flow into thedroplet generation junction to generate droplets that comprisemicrocapsules, and (ii) subjects the droplets to flow into the thirdchannel segment such that at least 75% of the droplets comprise at leastone microcapsule and fewer than 25% of the droplets comprise more thanone microcapsule.
 29. A microfluidic system, comprising: (a) amicrofluidic channel network, comprising: (i) a first channel segmentcoupled to a source of a first aqueous fluid that comprises a suspensionof microcapsules; (ii) at least one second channel segment coupled to asource of a second aqueous fluid, the first and second channel segmentsin fluid communication at a first junction that brings the first aqueousfluid in contact with the second aqueous fluid; (iii) a third channelsegment coupled to the first junction and intersecting at least onefourth channel segment at a second junction, the at least one fourthchannel segment being coupled to a source of a fluid that is immisciblewith the first and second aqueous fluids, and wherein the secondjunction partitions the first and second aqueous fluids into dropletswithin the fluid; and (b) a flow control system operably coupled to themicrofluidic channel network that subjects the first, second and thirdfluids to flow through the microfluidic channel network to form dropletscomprising the first and second aqueous fluids in the fluid, at afrequency of at least 50 Hz and that varies less than 20%. 30.(canceled)